Human interferon alpha comprises a group of secretory proteins with anti-viral, anti-proliferative and immunoregulatory activities (Pestka et al., 1987). Type I interferons, comprising the interferon alpha (IFN-α) family have specific effects on gene transcription and translation. They are a part of innate immunity, and represent the first barrier against virus infection. Interferon alpha also blocks cell growth. Moreover, it regulates the immune system, both by regulation of HLA expression and direct activation of several classes of killer cells. The interferons therefore represent tremendous clinical potential.
The human interferon alpha proteins are encoded by a family of thirteen genes on human chromosome 9. Eleven interferon alpha proteins have been identified from culture medium supernatants of Sendai virus-induced human leukocytes (Nyman et al., 1998; Adolf et al., 1990; Pohl et al., 1994). The different gene products are designated as subtypes, some of which represent allelic variants (Golovleva et al., 1996). Two of the genes, IFNA1 and IFNA13 have identical coding sequences and produce a single protein species, but the other interferon alpha subtype proteins differ at the protein level by as much as 22 percent (Adolf et al., 1990).
The interferons have therapeutic activity against a wide variety of malignant and viral diseases, but in general, the therapeutic and clinical application of natural interferon alpha has been slow due to both the high cost of obtaining human interferon alpha and the difficulties in purifying the product. Recombinant interferons can be produced on a large scale by using genetic engineering. Such recombinant interferons, however, comprise just a single subtype (for example, Schering Plough's Intron A (IFN-α2b) or Roche's Roferon (IFN-α2a)). These recombinant, or synthetic forms lack any post-translational modification, and this may limit their biological activity. Furthermore, production of neutralising antibodies in patients receiving the recombinant forms may limit the effectiveness of the treatment (Lok et al., 1990; Jacobs et al., 1988; Weck et al., 1989). IFN-α derived from human leukocyte preparations does not, however, elicit such a response (von Wussow et al., 1987; Liao et al., 1992; Antonelli et al., 1991) and this reported difference represents a significant advantage for the use of the ‘natural’ forms of interferon alpha.
Patients who have neutralising antibodies, and therefore have to stop treatment with recombinant interferon alpha, can be treated successfully with natural (leukocyte) interferon alpha (von Wussow et al. 1991; Merup et al. 1994; Berg et al. 2001).
The general method for purification of interferon alpha from human leukocytes is typically derived from the ‘Cantell’ method described in 1981 by Kari Cantell and others, involving sequential protein precipitation. However, this process provides only a partial purification method in that the resulting preparation is only 1 percent pure for interferon alpha. Further processing is required to produce the purity of product necessary for large-scale production for clinical trials.
In manufacturing a multi-component product such as human leukocyte-derived interferon alpha for human clinical use, it is imperative that the number and proportion of interferon alpha subtype proteins produced should remain consistent through different batches. The manufacturers must have the required degree of control over the process to produce a consistently high quality product. One way in which this may be achieved is through the use of product-specific antibody ligands in the purification process.
European Patent Application No EP 0,478,659A (BioNative AB, Sweden) describes the use of a process to purify human leukocyte-derived interferon alpha, one of the steps comprising the use of polyclonal antibodies specific for interferon alpha obtained by immunisation of goats with a recombinant interferon alpha. The polyclonal antibodies are conjugated to an immunoaffinity column matrix, and consistently recognise the 6 major interferon alpha subtype proteins which can be detected and which are characteristic of the commercially available multi-subtype interferon product Multiferon™ (formerly called Interferon Alfanative™, Viragen Inc.).
U.S. Pat. No. 5,240,864 (Koga et al., JCR Pharmaceuticals Co., Ltd.) describes the use of polyclonal antibodies which recognise all interferon alpha subtype proteins, and the further separation of the polyclonal mixture to produce antibodies specific for each subtype.
Monoclonal antibodies have proven to be an invaluable tool for the characterisation, quantitation and purification of macromolecular antigens and have revolutionised the large-scale manufacture of many proteins, giving far greater purity, improved production efficiencies and, therefore, lower manufacturing costs.
The interferon alpha proteins represent an interesting case study. On one hand, the 80-90 percent amino acid homology includes interferon alpha subtype proteins that are sufficiently dissimilar to cause difficulties in producing monoclonal antibodies which would recognise all natural interferon alpha proteins in consistent proportions. However, at the same time, many subtype proteins show a sufficiently close degree of similarity, to create problems in obtaining interferon alpha protein-specific monoclonal antibodies (Berg, 1984; Alkan & Braun, 1986).
There are many reports of interferon alpha subtype-specific monoclonal antibodies being used in purification processes. U.S. Pat. No. 4,973,556 (Bove et al., Schering Corporation) describes the use of hybridomas and resulting monoclonal antibodies specific for the interferon alpha 2 subtype. The monoclonal antibodies produced by Secher and Burke (1980), and the corresponding U.S. Pat. No. 4,423,147, describe the preparation of a monoclonal antibody which is specific only to one subtype. Staehelin et al., 1981a and b, produced thirteen different hybridomas, each specific for a particular interferon alpha subtype. A number of the subtype-specific antibodies produced from the hybridomas were immobilised by conjugation to a solid support and shown to be able to recognise the corresponding interferon alpha subtype proteins in human leukocyte interferon alpha. Furthermore, the Finnish Red Cross utilise two monoclonal antibodies which recognise all nine interferon alpha subtype proteins in the manufacture of a multi-subtype human leukocyte interferon alpha (Tolo et al., 2001 and the corresponding International PCT Patent Application Publication No WO 99/64440).
It would be desirable to produce and utilise a monoclonal antibody having a broad spectrum of reactivity when developing a large-scale process for purification of human leukocyte interferon alpha proteins. To maintain a high degree of quality in a product destined for human use, it is necessary to demonstrate that a process is robustly consistent: in this case, this refers to the proportion of each interferon alpha protein measured in the final product. Furthermore, the use of one monoclonal antibody which recognises a broad range of interferon alpha subtype proteins would be economically advantageous: this is a crucial point to consider in large-scale production. It would therefore be desirable to utilise just one monoclonal antibody in the large-scale immunopurification of a multi-subtype interferon alpha destined for human use.
Most monoclonal antibodies recognise only a certain number of interferon alpha proteins. U.S. Pat. No. 5,503,828 (Testa et al. Interferon Sciences), describes a process for the large scale purification of interferon alpha using a monoclonal antibody known as NK-2, which recognises at least seven interferon alpha proteins, specifically subtypes α2, α4, α7, α8, α17 and α21, but not α1 which is the most abundant interferon alpha subtype protein in the naturally derived multi-subtype interferon alpha product Multiferon™. A monoclonal antibody with reactivity to a different set of interferon alpha proteins has also been reported (Tsukui et al., 1986). This monoclonal antibody, known as HT-1, recognised interferon proteins α1, α2, α4 and α6. Some monoclonal antibodies have been produced which can reportedly recognise many interferon alpha proteins. Berg (1984), and corresponding U.S. Pat. No. 4,902,618 (Berg, Wadley Technologies Inc.) describes the production and use of a hybridoma cell line which produces a monoclonal antibody which recognises twelve subtypes of interferon alpha.
Monoclonal Antibody 3-A3-2 and Multiferon™
The natural multi-subtype interferon alpha product Multiferon™ (Viragen Inc., Florida, USA) is manufactured from human leukocytes stimulated with Sendai virus. The culture is typically purified according to the process described in U.S. Pat. No. 5,391,713 (BioNative AB). This production method depends on the use of polyclonal antibodies for interferon alpha production. Multiferon™ is composed of at least 6 major interferon alpha proteins: predominantly α1, with lesser amounts of proteins α2, α8, α10, α14 and α21. Table 1 lists the prescribed proportions of each interferon alpha protein found in the final Multiferon™ product.
For proteins such as interferon alpha, which are on the whole expensive to manufacture, it would be desirable to be able to use a monoclonal antibody to provide an interferon composition with greater purity, stability and consistency that the equivalent composition derived from a production process which uses a polyclonal antibody mixture.
The present inventors have surprisingly identified that when the monoclonal antibody 3-A3-2 is used in a process for the isolation and purification of human interferon alpha subtypes, the resulting interferon composition is identical in terms of interferon alpha subtype composition and proportions, to the interferon alpha subtype mix present in the natural multi-subtype interferon alpha product commercially available under the name Multiferon™. This composition was hitherto produced using goat derived polyclonal antibodies as described in U.S. Pat. No. 5,391,713. This observation is highly unexpected, as it could not have been predicted based on prior knowledge of the binding specificity of monoclonal antibody 3-A3-2.